Molecular Mechanisms of Apical Membrane Traffic in Epithelial Cells

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DESCRIPTION (provided by applicant): Epithelial cells establish and maintain the polarized distribution of membrane proteins and lipids by continuously sorting them during biogenesis and endocytic cycles. Protein targeting is critical for most epithelial functions. Consequently, its defects are known to lead to a variety of renal and digestive disorders. The main goal of my research is to investigate the mechanisms that regulate Rab 11-dependent apical protein targeting in epithelial cells. Targeting of proteins is achieved through the use of transport vesicles/tubules that fuse with specific target compartments. The vesicle protein, Rab 11, has been shown to be a key molecule involved in apical membrane traffic and may even function as a vesicle "address" tag. In general, Rabs function by recruiting effector proteins to transport vesicles. Recent studies have identified several novel Rab 11-binding proteins, including Rip 11, FIP2, and Eferin/FIP3, also known as FIPs. Based on my published and preliminary data, I hypothesize that (i) Rab 11/Rip11 complexes regulate apical membrane traffic by forming "targeting patches" on transport vesicles, (ii) Rab 11/Rip 11 "targeting patch" is regulated by Rip l 1 phosphorylation, and (iii) Rab 11/Rip 11 complex function by recruiting additional proteins, such as kinesin II to transport vesicles. Four specific aims are designed to further analyze the structure and function of Rab 11/effector complexes. First, the structural basis for Rab 11 interactions with Rip 11 will be investigated using domain mapping and X-ray crystallography. Second, we will use the structural information generated by Aim #1 to determine the role of Rip 11 as well as other FIPs in apical and basolateral targeting using in in vivo transport assays. Third, the role of Rip11 phosphorylation in polarized epithelial traffic will be investigated. Forth, identification and characterization of the proteins interacting with Rab 11/Rip 11 complexes. Characterization of Rab 11/Rip11 complex will provide critical mechanistic information for understanding protein targeting in epithelial cells.